Role of lipocalin 2 in intraventricular haemoglobin-induced brain injury.

OBJECTIVE: Our recent studies have shown that blood components, including haemoglobin and iron, contribute to hydrocephalus development and brain injury after intraventricular haemorrhage (IVH). The current study investigated the role of lipocalin 2 (LCN2), a protein involved in iron handling, in the ventricular dilation and neuroinflammation caused by brain injury in a mouse model of IVH. DESIGN: Female wild-type (WT) C57BL/6 mice and LCN2-deficient (LCN2-/-) mice had an intraventricular injection of haemoglobin, and control mice received an equivalent amount of saline. MRI was performed presurgery and postsurgery to measure ventricular volume and the brains were used for either immunohistochemistry or western blot. RESULTS: Ventricular dilation was observed in WT mice at 24 h after haemoglobin (25 mg/mL, 20 µL) injection (12.5±2.4 vs 8.6±1.5 mm3 in the control, p<0.01). Western blotting showed that LCN2 was significantly upregulated in the periventricular area (p<0.01). LCN2 was mainly expressed in astrocytes, whereas the LCN2 receptor was detected in astrocytes, microglia/macrophages and neurons. Haemoglobin-induced ventricle dilation and glia activation were less in LCN2-/- mice (p<0.01). Injection of high-dose haemoglobin (50 mg/mL) resulted in lower mortality in LCN2-/- mice (27% vs 86% in WT; p<0.05). CONCLUSIONS: Intraventricular haemoglobin caused LCN2 upregulation and ventricular dilation. Haemoglobin resulted in lower mortality and less ventricular dilation in LCN2-/- mice. These results suggest that LCN2 has a role in haemoglobin-induced brain injury and may be a therapeutic target for IVH.

Hyperbaric oxygen therapy and preconditioning for ischemic and hemorrhagic stroke.

To date, the therapeutic methods for ischemic and hemorrhagic stroke are still limited. The lack of oxygen supply is critical for brain injury following stroke. Hyperbaric oxygen (HBO), an approach through a process in which patients breathe in 100% pure oxygen at over 101 kPa, has been shown to facilitate oxygen delivery and increase oxygen supply. Hence, HBO possesses the potentials to produce beneficial effects on stroke. Actually, accumulated basic and clinical evidences have demonstrated that HBO therapy and preconditioning could induce neuroprotective functions via different mechanisms. Nevertheless, the lack of clinical translational study limits the application of HBO. More translational studies and clinical trials are needed in the future to develop effective HBO protocols.

Iron accumulation and DNA damage in a pig model of intracerebral hemorrhage.

Cerebral iron overload causes brain injury after intracerebral hemorrhage (ICH) in rats and pigs. The current study examined whether an iron chelator, deferoxamine, can reduce ICH-induced DNA damage in pigs. Pigs received an injection of autologous blood into the right frontal lobe. Deferoxamine (50 mg/kg, i.m.) or vehicle was given 2 h after ICH and then every 12 h up to 7 days. Animals were killed at day 3 or day 7 after ICH to examine iron accumulation and DNA damage. We found that ICH resulted in the development of a reddish perihematomal zone, with iron accumulation and DNA damage within that zone. Deferoxamine treatment reduced the perihematomal reddish zone, and the number of Perls' (p<0.01) and TUNEL (p<0.01) positive cells. In conclusion, iron accumulates in the perihematomal zone and causes DNA damage. Systemic deferoxamine treatment reduces ICH-induced iron overload and DNA damage in pigs.

Thrombin preconditioning reduces iron-induced brain swelling and brain atrophy.

Cerebral preconditioning with a low dose of thrombin attenuates brain edema induced by intracerebral hemorrhage (ICH), a large dose of thrombin or iron. This study examined whether or not thrombin preconditioning (TPC) reduces neuronal death and brain atrophy caused by iron. The right hippocampus of rats was pretreated with or without thrombin, and iron was then injected into the same location 3 days later. Rats were killed at 1 day or 7 days after iron injection, and the brains were used for histology. We found that TPC reduced neuronal death and brain swelling in the hippocampus 1 day after iron injection, and hippocampal atrophy 7 days later. Western blots showed that thrombin activates p44/42 mitogen-activated protein kinase (p44/42 MAPK) and 70-kDa ribosomal protein S6 kinase (p70 S6K). Our results indicate that TPC reduction of iron-induced neuronal death may be through the p44/42 MAPK /p70 S6K signal transduction pathway.

Effects of progesterone and testosterone on ICH-induced brain injury in rats.

Studies have shown that progesterone reduces brain injury, whereas testosterone increases lesion size after ischemic stroke. This study examined the effects of progesterone and testosterone on intracerebral hemorrhage (ICH)-induced brain injury. Male Sprague-Dawley rats received an injection of 100 µL autologous whole blood into the right basal ganglia. Progesterone (16 mg/kg), testosterone (15 mg/kg) or vehicle was given intraperitoneally 2 h after ICH. Behavioral tests were performed, and the rats were killed after 24 h for brain edema measurement. Perihematomal brain edema was reduced in progesterone-treated rats compared to vehicle-treated rats (p<0.05). Progesterone also improved functional outcome following ICH (p<0.05). Testosterone treatment did not affect perihematomal edema formation, but resulted in lower forelimb placing score (p<0.05). In conclusion, progesterone can reduce brain edema and improve functional outcome, whereas testosterone may have a deleterious effect after ICH in male rats.

Preconditioning with hyperbaric oxygen attenuates brain edema after experimental intracerebral hemorrhage.

OBJECT: Preconditioning with hyperbaric oxygen (HBO2) reduces ischemic brain damage. Activation of p44/42 mitogen-activated protein kinases (p44/42 MAPK) has been associated with preconditioning-induced brain ischemic tolerance. This study investigated if preconditioning with HBO2 protects against intracerebral hemorrhage (ICH)-induced brain edema formation and examined the role of p44/42 MAPK in such protection. METHODS: The study had three experimental groups. In Group 1, Sprague-Dawley rats received two, three, or five consecutive sessions of preconditioning with HBO2 (3 ata, 100% oxygen, 1 hour daily). Twenty-four hours after preconditioning with HBO2, rats received an infusion of autologous blood into the caudate. They were killed 1 or 3 days later for brain edema measurement. Rats in Group 2 received either five sessions of preconditioning with HBO2 or control pretreatment and were killed 24 hours later for Western blot and immunohistochemical analyses. In Group 3, rats received an intracaudate injection of PD098059 (an inhibitor of p44/42 MAPK activation) before the first of five sessions of preconditioning with HBO2. Twenty-four hours after the final preconditioning with HBO2, rats received an intracaudate blood infusion. Brain water content was measured 24 hours after ICH. RESULTS: Fewer than five sessions of preconditioning with HBO2 did not significantly attenuate brain edema after ICH. Five sessions of preconditioning with HBO2 reduced perihematomal edema 24 and 72 hours after ICH (p < 0.05). Strong p44/42 MAPK immunoreactivity was detected in the basal ganglia 24 hours after preconditioning with HBO2. Intracaudate infusion of PD098059 abolished HBO2 preconditioning-induced protection against ICH-induced brain edema formation. CONCLUSIONS: Preconditioning with HBO2 protects against brain edema formation following ICH. Activation of the p44/42 MAPK pathway contributes to that protection. Preconditioning with HBO2 may be a way of limiting brain injury during invasive neurosurgical procedures that cause bleeding.

Hyperbaric oxygen-induced attenuation of hemorrhagic transformation after experimental focal transient cerebral ischemia.

BACKGROUND AND PURPOSE: An increased risk of hemorrhagic transformation is a major factor limiting the use of tissue plasminogen activator for stroke. Increased hemorrhagic transformation is also found in animals undergoing transient focal cerebral ischemia with hyperglycemia; this study examined whether hyperbaric oxygen (HBO) could reduce such hemorrhagic transformation in a rat model. METHODS: Rats received an injection of 50% glucose (6 mL/kg intraperitoneally) and had a middle cerebral artery occlusion 10 minutes later. Rats were treated with HBO (3 ATA for 1 hour) 30 minutes after middle cerebral artery occlusion. Control rats received normobaric room air. Rats underwent reperfusion 2 hours after middle cerebral artery occlusion. Blood-brain barrier permeability (Evans blue), hemorrhagic transformation (hemoglobin content), brain edema, infarct volume, and mortality were measured. RESULTS: HBO treatment reduced Evans blue leakage in the ipsilateral hemisphere (28.4+/-3.5 versus 71.8+/-13.1 microg/g in control group, P<0.01) 2 hours after reperfusion and hemorrhagic transformation (0.13+/-0.13 versus 0.31+/-0.28 mg hemoglobin in the control group, P<0.05) 22 hours later. Mortality was less in the HBO group (4% versus 27% in controls, P<0.05). Mean infarct volume and swelling in the caudate were also less in HBO-treated rats (P<0.05), but HBO failed to reduce brain water content in the ipsilateral hemisphere (P>0.05). CONCLUSIONS: Early intraischemic HBO treatment reduces the blood-brain barrier disruption, hemorrhagic transformation, and mortality after focal cerebral ischemia suggesting that HBO could be used to reduce hemorrhagic conversion in patients with stroke.

Nestin expression after experimental intracerebral hemorrhage.

The current study examines nestin expression after intracerebral hemorrhage (ICH), the role of different blood components in nestin upregulation, and the possibility that low doses of thrombin that induce tolerance to brain injury (thrombin preconditioning) might also induce nestin expression. Adult male Sprague-Dawley rats received an intracaudate injection of either whole blood, thrombin (1 or 5 U) or red blood cells (RBCs). Animals were sacrificed for single and double labeling immunohistochemistry to identify which cells express nestin, and for Western blotting to quantify nestin expression. By immunohistochemistry, nestin immunoreactivity was present in large numbers of astrocytes, surrounding the hematoma from day 3 to 1 week after ICH. After 2 weeks, nestin immunoreactivity was co-localized with a neuronal marker (neuronal specific enolase). By Western blot analysis, nestin was strongly expressed at day 3 (P<0.01) and 1 week (P<0.01), and expression persisted for at least 1 month (P<0.05). Intracerebral injection of thrombin or lysed RBCs resulted in a marked increase in nestin expression. Interestingly, injection of a low dose of thrombin that induces brain tolerance also upregulated nestin. The ICH-induced nestin expression in astrocytes may reflect an early response of these cells to injury, while the delayed expression in neurons might be a part of the adaptative response to injury perhaps leading to recovery of function. Nestin induction by a low dose of thrombin suggests that specific receptor-mediated pathways are involved in inducing nestin expression and that nestin may play a role in thrombin preconditioning.

Deferoxamine-induced attenuation of brain edema and neurological deficits in a rat model of intracerebral hemorrhage.

OBJECT: In the authors' previous studies they found that brain iron accumulation and oxidative stress contribute to secondary brain damage after intracerebral hemorrhage (ICH). In the present study they investigated whether deferoxamine, an iron chelator, can reduce ICH-induced brain injury. METHODS: Male Sprague-Dawley rats received an infusion of 100 microl of autologous whole blood into the right basal ganglia and were killed 1, 3, or 7 days thereafter. Iron distribution was examined histochemically (enhanced Perl reaction). The effects of deferoxamine on ICH-induced brain injury were examined by measuring brain edema and neurological deficits. Apurinic/apyrimidinic endonuclease/redox effector factor-1 (APE/Ref-1), a repair mechanism for DNA oxidative damage, was quantitated by Western blot analysis. Iron accumulation was observed in the perihematoma zone beginning 1 day after ICH. Deferoxamine attenuated brain edema, neurological deficits, and ICH-induced changes in APE/Ref-1. CONCLUSIONS: Deferoxamine and other iron chelators may be potential therapeutic agents for treating ICH. They may act by reducing the oxidative stress caused by the release of iron from the hematoma.

Delayed argatroban treatment reduces edema in a rat model of intracerebral hemorrhage.

BACKGROUND AND PURPOSE: Studies indicate that thrombin plays an important role in intracerebral hemorrhage (ICH)-induced edema formation. Although thrombin is produced as the blood clots, it may be bound to fibrin and only gradually released from the clot. The time window for administration of a thrombin inhibitor to reduce ICH-induced edema is unknown. Whether this time window extends beyond the period when a thrombin inhibitor might exacerbate rebleeding is also unknown. METHODS: This study examines (1) whether argatroban, an inhibitor of both free and fibrin-bound thrombin, can reduce edema formation after intracerebral infusion of 100 micro L of blood in the rat; (2) the therapeutic time window for argatroban; and (3) whether argatroban promotes rebleeding in a model in which ICH was induced by intracerebral injection of collagenase. RESULTS: Intracerebral infusion of blood caused a marked increase in perihematomal water content. Intracerebral injection of argatroban 3 hours after ICH caused a significant reduction in edema measured at 48 hours (80.9+/-1.0% versus 82.6+/-0.8%; P<0.01). The systemic administration of high-dose argatroban (0.9 mg/h) starting 6 hours after ICH also significantly reduced edema (80.3+/-1.1% versus 82.0+/-1.3% in vehicle controls; P<0.05). There was no protection when the onset of argatroban administration was delayed to 24 hours after ICH or if a lower dose of argatroban (0.3 mg/h) was used. Argatroban did not increase collagenase-induced hematoma volume when given into the clot after 3 hours or given systemically at 6 hours. CONCLUSIONS: Our data suggest that argatroban may be an effective therapy for ICH-induced edema.